ABSTRACT
To determine the activity of matrix metalloproteinases (MMP), especially MMP-2 and MMP-9, which play an important role in ischemic stroke and intracerebral hemorrhage, we adapted a simple and rapid method for localizing gelatinase activity to a gelatin film in situ-overlay technique previously used in cancer research. Ten micrometer cryosections of rat brain from controls and animals subjected to 3 h of ischemia and 48 h of reperfusion (suture model for transient cerebral ischemia) were used. After thawing, a gelatin film with a polyester base was put on the slide, incubated for 24 h at 37 degrees C, stained with Ponceau S, and then discolored in bi-distilled water. Non-staining areas on the film corresponded to lysis zones, caused by activated MMPs. This was proven by MMP incubation at various concentrations on the plain gelatin film and pretreatment with EDTA (an MMP inhibitor), which prevents lysis zones in normal and ischemic brains. As confirmatory tests, SDS-PAGE zymography was used to define MMP activity, and also MMP-2 immunohistochemistry to detect the possibly cellular origin of MMPs. Normal rat brain exhibited a low background activity, which was visible as a light halo-like lysis zone over and around the brain. Areas in normal brain with medium MMP activity were within the white matter (corpus callosum, anterior commissure, and cerebellum). Ischemic brain exhibited high activity lysis zones within the infarcted area (detected by microtubuli associated protein-2 staining). These zones consisted of microscopically small lysis holes with a diameter of about 10-20 microm. Immunohistochemistry showed that especially microvessels expressed MMP antigen. SDS-PAGE zymography differentiated between a high level of activated MMPs in the ischemic area and a low level in the non-ischemic basal ganglia. The gelatin film in situ-overlay technique is able to localize MMP activity in ischemic rat brain tissue on a microscopic level.
Subject(s)
Brain Ischemia/enzymology , Brain/enzymology , Gelatin , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Animals , Brain/pathology , Brain Ischemia/pathology , Enzyme Activation , Immunohistochemistry , Male , Matrix Metalloproteinase 2/analysis , Matrix Metalloproteinase 9/analysis , Rats , Rats, WistarABSTRACT
A whole cell lysate of human cells was separated into 80 fractions according to the pI of proteins using free flow isoelectric focusing with carrier ampholytes. The resolution of the process was highly reproducible, with an overlap of fractions of less than 30%. A protein of a faint silver stained spot in two-dimensional gel electrophoresis (2-DE) could be enriched, yielding a Coomassie blue stained spot which could be further characterized by protein chemical methods. The enrichment of less abundant proteins from a complex crude cell extract was found to be a suitable tool for sample preparation and enrichment before applying proteins to 2-DE and reversed-phase high performance liquid chromatography.
Subject(s)
Isoelectric Focusing/methods , Proteins/analysis , Buffers , Cell Line , Chromatography, High Pressure Liquid , Electrophoresis, Gel, Two-Dimensional , HeLa Cells , Humans , Hydrogen-Ion Concentration , Reproducibility of ResultsABSTRACT
The master two-dimensional gel database (D. Burggraf et al., Electrophoresis 1992, 13, 729-732) [1] of common human proteins has been expanded to include detailed protein characteristics. Human cellular proteins from 5 cell lines and different cell organelles representing various tissues (muscle, nervous, connective, epithelial blood) and germ layers (ectoderm, endoderm and mesoderm), were separated by two-dimensional gel electrophoresis (2-DE). According to a recently developed algorithm, master gels of these different cells were established by computer-aided image processing. An expanded map with protein-chemical information of the polypeptides common to all human cells is shown. The synthetic, common human protein-map represents 856 spots resolved with an accuracy of 4 cm/pI unit. The protein spots were characterized either by their isoelectric point, molecular mass, integrated intensity, background-corrected optical density, spot area, or cellular distribution. About 80 proteins were further characterized and identified by protein name, amino acid composition analysis, N-terminal sequencing, enzymatic digest and subsequent peptide sequencing. Additionally the proteins of the common human protein map were identified by Western blotting. Specific information regarding glycosylation and quantitation of expression levels after chemical, biological and mechanical stimulation is included in the database.
Subject(s)
Databases, Factual , Electrophoresis, Gel, Two-Dimensional , Proteins/chemistry , Amino Acid Sequence , Amino Acids/analysis , Gels , Glycosylation , HeLa Cells , Humans , Image Processing, Computer-Assisted , Molecular Sequence Data , Tumor Cells, CulturedABSTRACT
A protein pattern of common human proteins was constructed by comparing the two-dimensional gel electrophoresis (2-DE) protein patterns from five cell lines of different germ layers. Total cell lysate and the isolated and purified nuclei of each cell line were separated by parallel electrophoresis runs in a multiple casting system of highest reproducibility. The computerized image analysis of the digitized 2-DE gels revealed a master protein pattern for each cell line. By comparison of all master protein patterns a 2-DE protein map of only common human proteins was constructed as a basis for a new 2-DE database. In a first step we have started characterizing a number of spots by microsequencing, amino acid composition analysis, and mass spectroscopy.
